Lysis and reverse transcription for mrna quantification

ABSTRACT

The present invention is directed to a method for performing an RT-PCR for amplifying a target RNA including the steps of (i) cultivation of a population of adherent cells in a cell culture vessel (ii) lysis of the population of adherent cells which is supposed to contain the target RNA in the sample vessel with a lysis buffer comprising between 0.05 M and 1 M of a chaotropic agent (iii) adding reagents to the sample vessel which are necessary to perform a reverse transcription reaction such that the chaotropic agent is present in a concentration of about 10 to 60 mM in the sample vessel, and reverse transcribing the target RNA and (iv) amplifying the first strand cDNA by means of subjecting the sample to multiple cycles of a thermocycling protocol.

RELATED APPLICATIONS

This application is filed as a division of U.S. application Ser. No.13/008,949, filed Jan. 19, 2011, which was filed as a continuation ofPCT/EP2009/005516, filed Jul. 30, 2009, and which claims priority to EP08013816.7, filed Aug. 1, 2008.

FIELD OF THE INVENTION

The present invention provides a new method for gene expressionmonitoring by means of performing RT-PCR. More precisely, the presentinvention discloses a possibility to lyse adherent cells in the vial inwhich they have been cultivated and subsequently, reverse transcribe theRNA contained in the lysate into single stranded cDNA in the same vial.

BACKGROUND OF THE INVENTION

Cells in a population are in many aspects unique in theircharacteristics, even in a seemingly homogenous culture or tissue. Geneexpression levels show large cell to cell variations, due to external(extrinsic) and internal (intrinsic) sources of factors Likewise, whenexposed to identical stimuli, cells often behave stochastically. Thismeans that data obtained from a population of cells cannot be assumed toreflect the behavior of the individual cell. It has been suggested thatcells can respond to stimuli by bursts in transcriptional activity andoperate as a binary switch; that is in an all-or-none fashion.

Usually, gene expression on the RNA level is monitored on a routinebasis by a multi-step procedure. First, the respective cellular sampleis removed from the culture vessel. In case of adherent cells harvestingmay be supported by trypsination (treatment with a Trypsin-EDTAsolution) in order to detach the adherent cell from the solid support.Secondly, the collected cells are pelleted and subjected to cell lysis.As a third step it is usually required to at least partially purify theRNA or mRNA that is present in the sample (EP 0 389 063). Afterwards, afirst strand cDNA synthesis step is performed with an RNA dependent DNApolymerase such as AMV (Roche Applied Science Cat. No: 11 495 062) orMMuLV (Roche 11 062 603) Reverse Transcriptase.

Subsequently, the amount of generated cDNA is quantified either by meansof quantitative PCR (Sagner, G., and Goldstein, C., BIOCHEMICA 3 (2001)15-17) or, alternatively by means of amplification and subsequenthybridization onto a DNA microarray (Kawasaki, E. S., Ann. N.Y. Acad.Sci. 1020 (2004) 92-100). In case of PCR, a one step RT-PCR may beperformed, characterized in that the first strand cDNA synthesis andsubsequent amplification are catalyzed by the same Polymerase such asT.th Polymerase (Roche Applied Science Cat. No. 11 480 014).

In traditional real time RT-PCR or qRT-PCR, RNA is first isolated fromcells in a time consuming procedure that can lead to a loss of material.Using the CellsDirect cDNA Synthesis System (Invitrogen Cat No.11737-030), the cells are lysed and the cDNA is generated from thelysate in a single tube with minimal handling and no sample loss. DNaseI is added to eliminate genomic DNA prior to first-strand synthesis.After synthesis, the first-strand cDNA can be transferred directly tothe qPCR reaction without intermediate organic extraction or ethanolprecipitation. This kit has been optimized for small cell samples,ranging from 10,000 cells down to a single cell. A similar protocol isdisclosed in (WO 08/135,197).

In this context, the technical problem underlying the present inventionwas to provide a high throughput method and a kit that allow for afurther simplified gene expression monitoring analysis protocol.

SUMMARY OF THE INVENTION

Thus, the present invention is directed to a method for performing anRT-PCR for amplifying a target RNA comprising the steps of

a) cultivation of a population of adherent cells in a cell culturevessel

b) lysis of said population of adherent cells which is supposed tocontain said target RNA in said sample vessel with a lysis buffercomprising between 0.05 M and 1 M of a chaotropic agent

c) adding reagents to said sample vessel which are necessary to performa reverse transcription reaction such that the said chaotropic agent ispresent in a concentration of about 10 to 60 mM in said sample vessel,and reverse transcribing said target RNA into first strand cDNA

d) amplifying said first strand cDNA by means of performing multiplecycles of a thermocycling protocol.

In one major embodiment, the step of amplifying is monitored in realtime.

Preferably, the chaotropic agent is Guanidine Thiocyanate.

Also preferably, the lysis buffer comprises between about 0.2 and 0.5 Mof the chaotropic agent.

During step c) of the inventive method, the chaotropic agent is presentin a concentration of about 30 to 50 mM and preferably about 40 mM.

Also preferably, step b) of the new method is performed in the presenceof a non ionic detergent which for example may be NP40. During step c),said non ionic detergent has a V/V of 0.1 to 2%.

Also preferably, step a) comprises the addition of a carbohydrate, whichmay be a sugar or a dextran.

In a particular embodiment, a DNAse is added between steps b) and c) orduring step c. Preferably, said DNAse is predominantly a double strandspecific DNAse, and preferably DNAse I or Shrimp Nuclease.

In another particular embodiment, which is not mutually exclusive withthe one disclosed above, Proteinase K is added either during step b) orprior to step c).

In a second aspect, the present invention also provides for a kitcomprising

a disposable for cultivating at least one cell sample,

a lysis buffer, and

a DNA polymerase comprising reverse transcriptase activity.

In addition, such a kit may further comprise at least one additionalcomponent selected from a group consisting of a non ionic detergent, acarbohydrate, a DNAse and a Protease.

BRIEF DESCRIPTION OF THE DRAWINGS

All figures comprise an upper panel representing a qPCR amplificationcurve and a lower panel representing a melting curve analysis.Amplification curves occurring on the more left side correspond toreplicate measurements, whereas amplification curves on the more rightside correspond to controls without addition of Transcriptor enzyme.Melting peaks on the right side correspond to specific amplificationproducts of replicates, whereas melting peaks on the more left sidecorrespond to primer dimer formation in controls without Transcriptorenzyme.

FIG. 1: qPCR and melting curve analysis of the ACTB expression in mouseastrocytes according to the present invention as embodied by example 1(1:1 sample dilution)

FIG. 2: qPCR and melting curve analysis of ACTB expression in mouseastrocytes according to the present invention as embodied by example 1(1:4 sample dilution)

FIG. 3: qPCR and melting curve analysis of TUBBS expression in mouseastrocytes according to the present invention as embodied by example 1(1:1 dilution)

FIG. 4: qPCR and melting curve analysis of ACTB expression in HeLA cellsaccording to the preset invention as embodied by example 1 (1:1dilution)

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, it is possible to perform a lysis ofadherent eukaryotic cells or even prokaryotic cells in a certaincultivation vessel and in the same cultivation vessel performing aReverse Transcriptase reaction in order to generate single strandedcDNA.

Thus, the present invention more precisely is directed to a method forperforming an RT-PCR for amplifying a target RNA comprising the steps of

a) cultivation of a population of adherent cells in a cell culturevessel

b) lysis of said population of adherent cells which is supposed tocontain said target RNA in said sample vessel with a lysis buffercomprising between 0.05 M and 1 M of a chaotropic agent.

c) adding all reagents to said sample vessel which are necessary toperform a reverse transcription reaction such that the said chaotropicagent is present in a concentration of about 10 to 60 mM in said samplevessel, and reverse transcribing said target RNA into first strand cDNA

d) amplifying said first strand cDNA by means of performing multiplecycles of thermocycling protocol.

Thus, cell cultivation, lysis, and the reverse transcription reaction,i.e. steps a) to c) are all performed in the same vessel. As aconsequence and in contrast to most prior art methods, there is nopurification of the lysate required prior to the performance of thereverse transcription reaction. In other words, step c) follows step b)without any intermediate purification step.

Step a), i.e. cultivation of a population of adherent cells in a cellculture vessel is defined as a step of growing a population of saidadherent cells under appropriate cultivation conditions over a definedperiod of time such that the cells have a possibility to divide and theoverall number of living cells increases at least by a factor of two.

Moreover, it is important to note that the protocols as used in the artrequire a trypsination step, which means that in order to detach theadherent cells from the solid support the cell culture is incubated withan appropriate buffer solution containing Trypsin-EDTA which arecommercially available (e.g. Invitrogen Cat. No: 25200 056, GenaxxonCat. No: 4260.0500). In contrast, the present invention does not requiredetachment of the cells from the solid support by trypsin, because thecells are directly lysed in situ.

Amplification according to step d) is usually performed in the form of aPCR reaction using a specific pair of amplification primers, which isdesigned to allow for the detection of a specific cDNA species.

The method according to the present invention may be used for a varietyof different qualitative and quantitative applications. In principle,any type of RNA may be transcribed and amplified. Most importantly, themethod according to the present invention is applicable for amplifyingand detecting mRNAs in a qualitative and quantitative manner. Thus, thepresent invention is particularly applicable for monitoring geneexpression.

In order to control the process of harvesting, cell lysis and reversetranscription, the samples may be spiked with a control RNA. The controlRNA preferably is an artificial RNA, which during the step of reversetranscription is transcribed into a cDNA that can be discriminated fromthe RNA of the sample. In case of using specific primers for the reversetranscription step, the artificial RNA may be derived from in vitrotranscription of a genetically engineered DNA template that eithercomprises an insertion or only partially represents the target sequencewhich shall become analyzed.

The biological sample preferably consists of adherent eukaryotic cells,i.e. the cells are cultivated and grow by being attached to a solidsupport that is part of a cultivation vessel. For the inventive method,any type of cultivation vessel can be used. Examples, which however, arenot limiting, the scope of the present invention are Petri dishes andcultivation bottles having an inner surface that is suited to be a solidsupport for the growth of adherent cells. These types of cultivationbottles allow for preparation of a specific type of cDNA in largequantities.

Other examples are microtiter plates in the 6-well, 24-well, 96-well,384-well, or 1536-well format as they are commonly used in the art. Incase the method according to the present invention is performed in suchmicrotiter plates, it is possible to cultivate, lyse and reversetranscribe multiple samples in parallel. More precisely, cellcultivation, cell lysis, dilution, any addition of additives and thereverse transcriptase reaction are carried out in the same reactionvessel. Therefore, the inventive method is particularly useful for highthroughput analyses of multiple samples of adherent cells within anautomated process. If the reaction vessels are arranged together in theform of a 24, 96, 384 or 1536 well microtiter plate according tostandards that are established in the art, the lysis reagent, thevarious additives and the reagents necessary for performing a ReverseTranscriptase reaction can be added to the samples by liquid handlingrobotic instruments.

Thus, in a particular aspect, the present invention is directed to amethod for performing multiple RT-PCR reactions for amplifying at leastone target RNA in parallel, comprising the steps of:

a) cultivation of multiple populations of adherent cells in separatewells of a microtiter plate

b) lysis of said multiple populations of adherent cells which aresupposed to contain said target RNA in said wells of said microtiterplate with a lysis buffer comprising between 0.05 M and 1 M of achaotropic agent in order to generate multiple samples

c) adding all reagents to each of said multiple samples in said wellswhich are necessary to perform a reverse transcription reaction suchthat the said chaotropic agent is present in a concentration of about 10to 60 mM in said wells of said microtiter plate, and reversetranscribing said target RNA present in each sample into first strandcDNA,

d) amplifying said first strand cDNA contained in each sample by meansof performing multiple cycles of a thermocycling protocol.

Within the scope of the invention, preferably 6, 24, 96, 384 or even1536 populations can be processed in parallel. Again, amplificationaccording to step d) is usually performed in the form of a PCR reactionusing a specific pair of amplification primers, which is designed toallow for the detection of a specific cDNA species. In one embodiment,the plurality of multiple wells may contain different cell populations,originating from different cell lines, or identical cell lines, whichhave been pre-treated under different conditions. In this case, themultiple PCR amplifications are performed using the same pair ofamplification primers in order to study the expression of a specificgene under different conditions.

Alternatively, the majority of wells may comprise the same type of cellpopulation originating from the same origin. In this case, multipledifferent pairs of amplification primers may be chosen in order to studythe expression of many different genes in parallel. However, a personskilled in the art will understand that both approaches as disclosed arenot mutually exclusive and, depending on the scientific question to beanalyzed, will be able to design an assay setup with an appropriatenumber of different cell types and an appropriate number of differentPCR primer pairs in order to achieve an optimal use of the completenumber of wells that are present in the respective microtiter plate.

In addition, the potential of such a parallel analysis can even beimproved by means of multiplex PCR, characterized in that multipleprimer pairs for the amplification of different species of cDNA are usedfor the same sample. In particular, such a setup is advantageous forrelative RT-PCR quantification experiments, characterized in that theexpression of a certain gene is monitored relatively to the expressionof a so called housekeeping gene, which is constitutively expressed atequal levels. Prominent examples of such housekeeping genes are thePBDG, GAPDH, and ACTB as well as 185 S and 26 S RNA genes.

According to the present invention, lysis of the adherent cells isachieved by means of directly adding a lysis buffer, while the cells arestill attached to a solid support surface. This is only possible if thelysis buffer comprises a chaotropic reagent. According to commonunderstanding in the art, a chaotropic reagent is a substance whichdisrupts the three dimensional structure in macromolecules such asproteins, DNA, or RNA and denatures them. Chaotropic agents interferewith stabilizing intramolecular interactions mediated by non-covalentforces such as hydrogen bonds, Van der Waals forces, and hydrophobiceffects. Chaotropic reagents include but are not limited to urea, somelithium salts such as lithium perchlorate and guanidinium salts such asguanidinium chloride. In the context of the present invention, theparticularly preferred agent is guanidine thiocyanate.

In order to enable the possibility to perform the reverse transcriptasereaction directly within the lysate without any intermediatepurification step, it is required to use only limited amounts of saidchaotropic agent for lysis of the cell sample, such that during step b),the activity of reverse transcriptase is not appreciably affected.Therefore, the lysis buffer added in step b) comprises preferablybetween about 0.2 and 0.5 M of the chaotropic agent. Furthermore, afteraddition of additional reagents at the beginning of step c) thechaotropic agent is present in a concentration of about 30 to 50 mM andpreferably about 40 mM.

In one embodiment, step b) of the present invention is performed in thepresence of a non ionic detergent such as NP40. Alternatively, step b)comprises the addition of a PCR compatible humectant, which results inan effective avoidance of evaporation effects, if samples with onlysmall volumes need to be processed. Preferably, a carbohydrate such as asugar or a dextran may be used as a humectants in the context of thepresent invention. If NP40 or another non ionic detergent is added, thenthe amount of detergent should be chosen in such a way that during stepc) said detergent is present in a V/V amount of 0.1 to 2%.

Cell harvesting and lysis according to the present invention may beperformed at various temperatures. Step b) of the inventive method isusually performed for at least 5 minutes i.e. between 16° C. and 24° C.The maximum time which is required for lysis under these circumstancesis about 30 minutes. Similarly, step b) can be performed for at least 10minutes even below ambient temperature, but above 5° C. The maximum timewhich is required for lysis under these circumstances is about 60minutes. These conditions are very favorable for the avoidance of anyevaporation effects, if samples with small volumes are processed. Italso eliminates the need for heating.

Yet, in order to accelerate and improve lysis of the cultivated adherentcells, the lysis buffer may be supplemented with Proteinase K. In thiscase, step b) is performed for at least 5 minutes at a temperaturebetween about 55° C. to 85° C. in the presence of Proteinase K inconcentrations of about 0.05 to 5 mg/ml and preferably 0.1-1 mg/ml.Optionally, said Proteinase K may be irreversibly inactivated by meansof subsequent incubation either between step b) and step c) or betweenstep c) and step d) for at least 5 minutes but usually not more than 30minutes at a temperature between about 80° C. to 90° C.

As according to the present invention, cell lysis and reversetranscription are performed in the same reaction vessel, it has beenproven to be highly advantageous if the genomic DNA that is contained inthe lysed cells can selectively be removed, while the cellular RNA ismaintained intact. The most effective possibility to achieve this effectis an enzymatic removal by means of including a DNAse digestion step.Thus, in one major embodiment of the present invention, step b) isperformed in the presence of a double strand specific DNAse. Preferably,such a DNAse is an exclusively double strand specific DNAs such as DNAseI (Roche Applied Science Cat. No. 04 716 728) or Shrimp DNAse (U.S. Pat.No. 6,541,204) at a concentration of about 0.1 Unit per 50 ti reaction(USB, Cat. No: 73814). However, since during step d) the single strandedcDNA is further subjected to a DNA Polymerase catalyzed amplificationreaction such as a PCR reaction, it is highly advantageous to inactivatesaid DNAse prior to the amplification reaction. Thus, for inactivationof DNAse activity, in a specific embodiment the sample is incubated forat least 5 minutes but not longer than 60 minutes between step b) andstep c) or step c) and step d) at a temperature between about 80° C. to90° C. Alternatively, if the DNAse is Shrimp DNAse, the denaturationduring the first cycle of the PCR reaction in step d) is usuallysufficient.

For the first strand cDNA synthesis, primers with an antisense sequenceare used. These primers are either specific primers, olig-dT primers,which bind to the poly-A-tail of an mRNA, or random primers such asrandom hexamer primers. For subsequent PCR, a sequence specific primerin sense orientation is used as a forward primer. The reverse primer isa specific primer which may be identical to the specific primer used inthe first strand cDNA synthesis reaction. Alternatively, the reverseprimer, which hybridizes to a sequence located upstream from the bindingside of the primer that has been used for the reverse transcriptasereaction.

The present invention is applicable for performing one step RT-PCR ofsequences of practicably any amplicon size up to 5 kb.

The present invention is particularly useful for performing 2-step RTPCR, i.e. in a first reaction, the RNA is reverse transcribed intosingle stranded cDNA. Examples for RNA dependent DNA polymerases thatcan be used for this step are AMV Reverse Transcriptase (Roche AppliedScience Cat. No. 11 495 062), MMuL V Reverse Transcriptase (RocheApplied Science Cat. No. 03 531 317). Subsequently, all reagents areadded that are required to amplify the generated single stranded cDNA bymeans of PCR, such as a thermostable DNA dependent DNA polymerases aswell as target specific forward and reverse amplification primers.

The inventive method can also be used for performing 1-step PCR,characterized in that all reagents and enzymes necessary for RT-PCR areadded in step c) prior to the reverse transcription. For example, theDNA polymerase of Carboxydothermus hydrogenoformans is capable ofperforming a 1-step PCR (Roche Applied science Catalog No. 12016338001).Alternatively, the one step RT-PCR method according to the presentinvention is performed using an enzyme mixture comprising a DNA-templatedependent thermostable DNA Polymerase capable of performing a PCRreaction and an RNA-template dependent DNA Polymerase capable ofperforming the reverse transcriptase step of the one step RT-PCRreaction such as AMV Reverse Transcriptase.

Usually, however, the material of the majority of cell culture vesselsused for cultivating adherent cells will not be thermostable, and therespective vials will not fit into a thermocycling instrument. Thus, fora 1-step RT-PCR protocol, after the step of reverse transcription, thesamples of the generated first strand cDNA are transferred from thecultivation vessels into reaction vessels that can be used inconjunction with a thermocycler instrument without an intermediateaddition of further reagents. Yet, if the material of the cell culturevessels used for cultivating said adherent cells is thermostable, thenstep d) may also be performed in said vessels. In particular, if thecell culture vessels are arranged in a microtiter plate format, thensaid microtiter plate can be transferred directly to thermocyclerinstruments that are configured to take up such plates.

In one major embodiment, the step of amplifying is monitored in realtime. Such a monitoring in real time is characterized in that theprogress of said one-step RT-PCR reaction is monitored in real time.Different detection formats are known in the art. The below mentioneddetection formats have been proven to be useful for RT-PCR and thusprovide an easy and straight forward possibility for gene expressionanalysis:

a) Taqman Hydrolysis Probe Format:

A single-stranded Hybridization Probe is labeled with two components.When the first component is excited with light of a suitable wavelength,the absorbed energy is transferred to the second component, theso-called quencher, according to the principle of fluorescence resonanceenergy transfer. During the annealing step of the PCR reaction, thehybridization probe binds to the target DNA and is degraded by the 5′-3′exonuclease activity of the Taq Polymerase during the subsequentelongation phase. As a result the excited fluorescent component and thequencher are spatially separated from one another and thus afluorescence emission of the first component can be measured. TaqManprobe assays are disclosed in detail in U.S. Pat. No. 5,210,015, U.S.Pat. No. 5,538,848, and U.S. Pat. No. 5,487,972. TaqMan hybridizationprobes and reagent mixtures are disclosed in U.S. Pat. No. 5,804,375.

b) Molecular Beacons:

These hybridization probes are also labeled with a first component andwith a quencher, the labels preferably being located at both ends of theprobe. As a result of the secondary structure of the probe, bothcomponents are in spatial vicinity in solution. After hybridization tothe target nucleic acids both components are separated from one anothersuch that after excitation with light of a suitable wavelength thefluorescence emission of the first component can be measured (U.S. Pat.No. 5,118,801).

c) FRET Hybridization Probes:

The FRET Hybridization Probe test format is especially useful for allkinds of homogenous hybridization assays (Matthews, J. A., and Kricka,L. J., Analytical Biochemistry 169 (1988) 1-25). It is characterized bytwo single-stranded hybridization probes which are used simultaneouslyand are complementary to adjacent sites of the same strand of theamplified target nucleic acid. Both probes are labeled with differentfluorescent components. When excited with light of a suitablewavelength, a first component transfers the absorbed energy to thesecond component according to the principle of fluorescence resonanceenergy transfer such that a fluorescence emission of the secondcomponent can be measured when both hybridization probes bind toadjacent positions of the target molecule to be detected. Alternativelyto monitoring the increase in fluorescence of the FRET acceptorcomponent, it is also possible to monitor fluorescence decrease of theFRET component as a quantitative measurement of hybridization event.

In particular, the FRET Hybridization Probe format may be used in realtime PCR, in order to detect the amplified target DNA. Among alldetection formats known in the art of real time PCR, theFRET-Hybridization Probe format has been proven to be highly sensitive,exact and reliable (WO 97/46707; WO 97/46712; WO 97/46714). As analternative to the usage of two FRET hybridization probes, it is alsopossible to use a fluorescent-labeled primer and only one labeledoligonucleotide probe (Bernard, P. S., et al., Analytical Biochemistry255 (1998) 101-107). In this regard, it may be chosen arbitrarily,whether the primer is labeled with the FRET donor or the FRET acceptorcompound.

d) SybrGreen Format

It is also within the scope of the invention, if real time PCR isperformed in the presence of an additive according to the invention incase the amplification product is detected using a double strandednucleic acid binding moiety. For example, the respective amplificationproduct can also be detected according to the invention by a fluorescentDNA binding dye which emits a corresponding fluorescence signal uponinteraction with the double-stranded nucleic acid after excitation withlight of a suitable wavelength. The dyes SybrGreenI and SybrGold(Molecular Probes) have proven to be particularly suitable for thisapplication. Intercalating dyes can alternatively be used. However, forthis format, in order to discriminate the different amplificationproducts, it is necessary to perform a respective melting curve analysis(U.S. Pat. No. 6,174,670).

Kits According to the Present Invention

In another aspect, the present invention also provides for a kitcomprising

(i) a disposable for cultivating at least one cell sample,

(ii) a lysis buffer, and

(iii) a DNA polymerase comprising reverse transcriptase activity(Reverse Transcriptase)

The disposable (i) may be any kind of disposable that is suited for thecultivation of at least one cell population. Preferably, such adisposable is a microtiter plate with preferably 6-, 24-, 96-, 384-, or1536 wells. In a specific embodiment, such a disposable is thermostableand compatible with a thermocycler instrument.

The lysis buffer (ii) is a buffer comprising between 0.05 M and 1 M of achaotropic agent. The agent itself is preferably guanidine thiocyanate.Optionally, the kit may contain Proteinase K within the lysis buffer ina concentration of about 0.05 to 5 mg/ml and preferably 0.1-1 mg/ml, oras a standalone reagent with an at least 5 fold higher stockconcentration. Also optionally, the kit may contain a dextran or a nonionic detergent, which is preferably NP, either within the lysis bufferin a V/V amount of 0.1 to 2%, or alternatively in a separate vial an atleast 5 fold higher stock concentration. Also optionally, the kit maycontain a predominantly double strand specific DNAse such as DNAse I orShrimp Nuclease in a separate vial at a concentration of at least 0.02unit.

The Reverse Transcriptase (iii) is either an exclusively RNA dependentDNA polymerase that such as AMV Reverse Transcriptase (Roche AppliedScience Cat. No. 11 495 062), MMuLV Reverse Transcriptase (Roche AppliedScience Cat. No. 011 062 603), and the recombinant Transcriptor ReverseTranscriptase (Roche Applied Science Cat. No. 03 531 317). Component(iii) may also be a 1-step RT PCR enzyme such as the DNA polymerase ofCarboxydothermus hydrogenoformans, or a mixture of a ReverseTranscriptase enzyme and a thermostable DNA dependent DNA polymerase.

A kit according to the present invention may contain a buffer,deoxynucleoside triphosphates that are required for a first strand cDNAsynthesis, as well as respective primers such as an olig-dT primer, arandom hexamer primer or even a specific primer. Together with thepolymerase, one, several or all of these reagents may be combined intoone vial as a respective master mix.

Furthermore, such a kit according to the present invention may comprisea thermostable DNA polymerase such as Taq Polymerase, and all otherreagents necessary for performing the amplification reaction, includingbut not limited to a buffer reagent, additional deoxynucleosidetriphosphates, and sequence specific amplification primers. In addition,the kit may comprise reagents necessary for detection of the ampliconduring qPCR such as at least one fluorescently labeled hybridizationprobe, or a double stranded fluorescent dye.

Example 1

Mouse astrocyte cells were seeded at two different concentrations (1:1,1:4) intoa 384-well cell culture plate with approximately 1000 or 250cells per vial and allowed to grow. After about 96h, the culture mediawas removed and the cells were washed with cold PBS. 2 μl of lysisbuffer was added and the cells were incubated for 10 minutes at roomtemperature. Composition of lysis buffer was as follows:

0.2 M guanidine thiocyanate

10 mg/μl polyinosinic acid

Transcriptor Rt reagents (Roche Applied Science Cat. No. 03 531 317)were added to a final RT volume of 20 μl. In No RT controls, the reversetranscriptase was omitted from the RT mix. The cell culture platecontaining the RT reactions was incubated according to the followingthermocycling protocol:

25° C. for 10 minutes

55° C. for 30 minutes

85° C. at 5 minutes

Chill on ice.

Subsequently, the cDNA was diluted to 50 μl in nuclease free water. 2 μlof cDNA was analyzed in a 10μl PCR using the LightCycler 480 SYBR GreenI Master pre-mix (Roche Applied Science Cat. No. 04 707 516) and primerssuitable for amplifying ACTB and 18S rRNA (TATAA Biocenter, endogenouscontrol gene panel). The expression of the ACTB and 18S rRNA genes wasquantified on a LightCycler 480 real-time PCR instrument (Roche AppliedScience Cat. No. 05 015 278) and Ct-values were determined according tothe manufacturer's instructions. Low Ct values correspond to a highdegree of expression.

TABLE 1 Ct values obtained for the 1:1 dilution Sample name ACTB - Ct18S rRNA - Ct Well 1 27.62 11.52 Well 2 27.23 11.49 Well 3 27.9 12.06Well 4 27.8 11.81 NoRT 1 36.95 32.56 NoRT 2 37.35 30.45

TABLE 2 Ct values obtained for the 1:4 dilution Sample name ACTB - Ct18S rRNA - Ct Well 1 26.82 10.56 Well 2 27.06 10.52 Well 3 28.85 11.7Well 4 26.47 10.06 NoRT 1 38.28 32.29 NoRT 2 38.18 32.5As can be deduced from the tables, the Ct values obtained for respectivequadruplicates are highly reproducible, indicating the high performancequality of the claimed invention.

In addition, results of qPCR and subsequent melting curve analysis forthe ACTB gene are shown in FIGS. 1 (1:1) and 2 (1:4).

Example 2

Mouse astrocyte cells were seeded into a 384-well cell culture platewith an average number of approximately 125 cells per vial and allowedto grow. After about 96h, the culture media was removed and the cellswere washed with cold PBS buffer. The PBS buffer was then replaced withRNA later (Ambion, Cat. No. 7020), and the cells were stored in thefridge for 11 days. The RNA later was then removed, and the cells werewashed once or twice with cold PBS. 4 μl of lysis buffer containing 0.2M guanidine thiocyanate and 5 ml/μl polyinosinic acid was added and thecells were incubated for 10 minutes at room temperature. Transcriptor RTreagents were added to a final RT volume of 20 μl. In No RT controls thereverse transcriptase was omitted from the RT mix. The cell cultureplate containing the RT reactions was incubated according to thefollowing protocol:

25° C. for 10 minutes

50° C. for 30 minutes

85° C. at 5 minutes

Chill on ice

The cDNA was diluted to 50 μl in nuclease free water. Analogous toexample 1, 2 μl of cDNA was analyzed in a 10 μl PCR using theLightCycler 480 SYBR Green I Master pre-mix. Real-time PCR measuring the18S rRNA, ACTB, GAPDH and TUBBS genes with appropriate primers (TATAABiocenter, endogenous control gene panel) was performed on a LightCycler480 real-time PCR instrument. Results of Ct values obtained are shown intable 3.

TABLE 3 ACTB - 18S rRNA - GAPDH - TUBB5 - Sample name Ct Ct Ct Ct 1 PBSwash well 1 26.14 10.95 24.05 23.96 1 PBS wash well 2 26.20 10.55 23.8923.85 1 PBS wash well 3 26.63 10.80 24.00 24.03 1 PBS wash NoRT 32.0628.19 28.74 40.00 2 PBS washes well 1 27.67 11.58 24.65 25.32 2 PBSwashes well 2 27.17 11.31 24.51 25.20 2 PBS washes well 3 27.13 11.3424.48 25.17 2 PBS washes NoRT 36.47 30.69 32.94 N/AAgain, the very similar Ct values obtained for the triplicates show thesuperior performance of the inventive method irrespective of the genethat is analyzed. Performance is also demonstrated in FIG. 3, whichshows the amplification and melting curves obtained for the TUBBS gene.

Example 3

Human cultured HeLa cells were seeded into a 96-well cell culture platewith an approximate number of 4000 cells per vial and allowed to grow.After about 48h, the culture media was removed and replaced with RNAlater, and the cells were stored in the fridge for 24 h. The RNA laterwas then removed, and the cells were washed with cold PBS. 12.5 μl oflysis buffer according to example 1 was added and the cells wereincubated for 10 minutes at room temperature. Transcriptor RT reagentswere added to a final RT volume of 50 μl. In No RT controls the reversetranscriptase was omitted from the RT mix. The cell culture platecontaining the RT reactions was incubated according to the followingprotocol:

25° C. for 10 minutes

50° C. for 30 minutes

85° C. at 5 minutes

Chill on ice

Analogous to example 1, 2 μl of cDNA was analyzed in a 10 μl PCRreaction using the LightCycler 480 SYBR Green I Master pre-mix.Real-time PCR measuring the ACTB, GAPDH, RPLP0 and HPRT1 genes withappropriate primers (TATAA Biocenter, endogenous control gene panel) wasperformed on a LightCycler 480 real-time PCR instrument. The Ct valuesobtained are shown in table 4.

TABLE 4 Sample name ACTB - Ct GAPDH - Ct RPLP0 - Ct HPRT1 - Ct Well 121.0 19.83 21.71 24.74 Well 2 21.71 21.63 23.57 26.09 Well 3 21.19 20.9522.86 25.88 Well 4 21.8 20.94 23.2 25.7 Well 5 23.03 22.0 24.49 26.7NoRT 40.0 N/A 40.0 N/A

The very similar Ct values obtained for the pentaplicates show thesuperior performance of the invented method irrespective of the celltype analyzed. Performance is also demonstrated in FIG. 4, which showsthe amplification and melting curves obtained for the ACTB gene.

What is claimed:
 1. A kit comprising: a disposable vessel suitable forcultivating at least one adherent cell sample; and a lysis buffercomprising a chaotropic agent; and reagents necessary to perform areverse-transcription reaction, said reagents comprising a DNApolymerase capable of reverse transcriptase activity.
 2. The kitaccording to claim 1, further comprising at least one additionalcomponent selected from the group consisting of a non-ionic detergent, acarbohydrate, a deoxyribonuclease (DNAse) and a protease.
 3. The kitaccording to claim 1, wherein the chaotropic agent is guanidinethiocyanate.
 4. The kit according to claim 1, wherein the lysis buffercomprises between 0.05 M and 1M of the chaotropic agent.
 5. The kitaccording to claim 2, wherein the lysis buffer comprises a non-ionicdetergent.
 6. The kit according to claim 5, wherein a total reactionvolume is equal to the volume of all kit components added to the vesseland the lysis buffer comprises non-ionic detergent in a concentrationsuch that non-ionic detergent is present in the total reaction volume ata concentration of between about 0.1 and about 2% V/V.
 7. The kitaccording to claim 2, wherein at least one additional componentcomprises DNAse.
 8. The kit according to claim 2, wherein at least oneadditional component comprises a protease.
 9. The kit according to claim8, wherein the protease comprises proteinase K.
 10. The kit according toclaim 3, wherein a total reaction volume is equal to the volume of allkit components added to the vessel and the lysis buffer compriseschaotropic agent in a concentration such that chaotropic agent ispresent in the total reaction volume at a concentration of between about10 and about 60 mM.
 11. The kit according to claim 10, wherein thechaotropic agent is guanidine thiocyanate.